PROJECT SUMMARY COPD is now the 3rd leading cause of death worldwide. Emphysema, characterized by airspace enlargement and impaired gas exchange, is a major subtype of COPD. The cumulative effects of cigarette smoke exposure (CS) and environmental toxins across the lifespan are important contributing factors to the development of emphysema. Yet very little is known about the underlying mechanisms, which limits therapies. Our overall goals are to understand the molecular mechanisms of how our lungs maintain normal structural integrity and how they protect against inhaled toxins, such as CS. We identified a critical role for an innate immune receptor, Toll-like receptor 4 (TLR4), in lung maintenance and in preventing inappropriate activation of aging and injury pathways. Unfortunately, age and CS, two of the most common risk factors for COPD, result in inadequate levels of TLR4, thus predisposing to emphysema. Our studies will fill current gaps in our understanding of how the immune system, in specific lung cells, can impact lung-protective responses even in the absence of infection, thereby offering potential new therapeutic targets. Using TLR4-deficiency and CS as clinically relevant experimental models of emphysema, we identified mechanistic roles for the senescence molecule, p16INK4A, and DNA-modifying enzymes, HDAC2, Dnmt3a and Tet2, in emphysema. In addition, we offer the first in vivo evidence that increased p16INK4A contributes to emphysema. Our overall hypothesis is that structural cell TLR4 is required to maintain normal lung integrity, at baseline and after CS, by inhibiting p16INK4A via HDAC2-Dnmt3a-Tet2-mediated mechanisms. We will use a combination of age- and CS- exposed human and mouse lungs/cells as well as innovative gene manipulations to test this hypothesis in the following Aims: 1) Identify mechanisms of decreased TLR4 expression with aging and CS and the contribution of TLR4 in specific structural cells to emphysema. 2) Determine the mechanisms of decreased HDAC2 in TLR4-deficient and CS-exposed cells. 3) Identify HDAC2 and Dnmt3a / Tet2 -mediated mechanisms of p16INK4A induction and the contribution of p16INK4A in specific structural cells to emphysema. Our studies will expand our basic understanding of the molecular drivers of emphysema, establish previously unrecognized interactions amongst innate immunity, senescence and DNA modifications and inform future preventative or therapeutic approaches to a range of age- and CS-related lung diseases.